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841 XTX202, a tumor-selective protein-engineered IL-2, exhibited enhanced anti-tumor activity in combination with checkpoint inhibition in mice
  1. Wilson Guzman,
  2. Uli Carl Bialucha,
  3. Hanumantha Rao Madala,
  4. Haley Duprey,
  5. Manoussa Fanny,
  6. Stephanie Hsiao,
  7. Parker Johnson,
  8. Jake Taylor,
  9. Rebekah O’Donnell,
  10. Magali Pederzoli-Ribeil,
  11. Benjamin Nicholson,
  12. Jennifer O’Neil and
  13. Caitlin O’Toole
  1. Xilio Therapeutics, Waltham, MA, United States


Background High-dose recombinant human interleukin-2 (IL-2, aldesleukin) is approved for the treatment of renal cell carcinoma (RCC) and melanoma based on durable responses. However, use of aldesleukin is limited due to treatment-related life-threatening toxicities. With the goal of overcoming these toxicities and improving the therapeutic index of IL-2, we employed protein engineering (Geographically Precise Solutions) to generate XTX202, a masked, non-alpha tumor-selective IL-2. XTX202 is designed to bias its binding towards beta/gamma IL-2 receptors and be pharmacologically inactive until unmasked by proteases that are enriched in the tumor microenvironment, resulting in activation and IL-2 signaling.

Methods The in vitro bioactivity of masked and proteolytically unmasked XTX202 was compared using STAT-5 phosphorylation in human CD8+T (CD8) and regulatory T (Treg) cells. Dissociated cells from primary human tumor tissues or patient plasma were incubated with XTX202, and % of cleaved XTX202 was measured by Western blot. Tumor growth was monitored after treatment with XTX202 alone and in combination with checkpoint inhibition in human FcRn transgenic mice bearing syngeneic MB49 tumors.

Results XTX202 has attenuated IL-2 receptor signaling in CD8 and Treg cells, as compared to its unmasked control XTX200. Upon activation by proteases, XTX202 had comparable activity in CD8 and Treg cells to that of its unmasked control XTX200. Unlike recombinant IL-2, unmasked XTX202 has similar potency on CD8 and Treg cells, rather than higher potency on Treg cells. XTX202 was cleaved by a broad range of human primary solid tumors (29-100% of samples tested were cleaving XTX202). XTX202 was not significantly activated by plasma from healthy control donors, and RCC patients. In vivo data indicated that single-agent dosing with XTX202 resulted in anti-tumor activity in the MB49 murine bladder carcinoma model. Combining XTX202 with checkpoint inhibition further enhanced the anti-tumor activity.

Conclusions XTX202, a tumor-selective IL-2, was proteolytically activated by a broad range of solid primary human tumors and minimally cleaved in plasma. In the syngeneic mouse bladder MB49 model, XTX202 as a single agent showed significant tumor-growth inhibition, and no evidence of toxicity or peripheral immune activation was observed, thus demonstrating tumor-selective activity in this model. The combination of XTX202 with immune checkpoint blockade demonstrated further enhancement in the anti-tumor activity in MB49 tumor bearing mice. Clinical testing of XTX202 is ongoing (NCT05052268).

Ethics Approval All animal procedures were either approved by an Institutional Animal Care and Use Committee and conducted in accordance with the National Institutes of Health

Guide for the Care and Use of Laboratory Animals.

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